Fermentation process for the preparation of L-amino acid using modified Escherichia coli wherein the ytfP-yjfA gene region is inactivated

ABSTRACT

The present invention relates to a fermentation process for the preparation of L-amino acids in which the following steps are carried out: (a) fermentation of the microorganisms of the family Enterobacteriaceae producing the desired L-amino acid, in which microorganisms&#39; open reading frames of yjfA and ytfP are individually or jointly inactivated by one or more methods of mutagenesis selected from the group consisting of deletion, insertional mutagenesis due to homologous recombination, and transition or tranversion mutagenesis with incorporation of a non-sense mutation in the yjfA and ytfP gene region; (b) concentration of the fermentation broth to eliminate water and increase the concentration of said L-amino acids; and (c) isolation of the L-amino acids.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 09/963,668,filed Sep. 27, 2001, now U.S. Pat. No. 6,916,637, which claims priorityto U.S. Provisional Patent Appl. No. 60/237,610, filed Oct. 4, 2000, andGerman Patent Application Nos. 100 48 605.3, 100 55 516, and 101 30192.8, filed Sep. 30, 2000, Nov. 9, 2000, and Jun. 22, 2001,respectively.

FIELD OF THE INVENTION

The present invention relates to a fermentation process for thepreparation of L-amino acids, especially L-threonine, using strains ofthe family Enterobacteriaceae in which at least the pckA gene isattenuated.

PRIOR ART

L-Amino acids are used in animal nutrition, in human medicine and in thepharmaceutical industry.

It is known to prepare L-amino acids by the fermentation of strains ofEnterobacteriaceae, especially Escherichia coli and Serratia marcescens.Because of their great importance, attempts are constantly being made toimprove the preparative processes. Improvements to the processes mayrelate to measures involving the fermentation technology, e.g. stirringand oxygen supply, or the composition of the nutrient media, e.g. thesugar concentration during fermentation, or the work-up to the productform, e.g. by ion exchange chromatography, or the intrinsic productivitycharacteristics of the microorganism itself.

The productivity characteristics of these microorganisms are improved byusing methods of mutagenesis, selection and mutant choice to givestrains which are resistant to antimetabolites, e.g. the threonineanalogue α-amino-β-hydroxyvaleric acid (AHV) or auxotrophic formetabolites of regulatory significance, and produce L-amino acids, e.g.L-threonine.

Methods of recombinant DNA technology have also been used for some yearsto improve L-amino acid-producing strains of the familyEnterobacteriaceae by amplifying individual amino acid biosynthesisgenes and studying the effect on production.

OBJECT OF THE INVENTION

The object which the inventors set themselves was to provide novelprocedures for improving the preparation of L-amino acids, especiallyL-threonine, by fermentation.

SUMMARY OF THE INVENTION

The invention provides a fermentation process for the preparation ofL-amino acids, especially L-threonine, using microorganisms of thefamily Enterobacteriaceae which, in particular, already produceL-threonine and in which the nucleotide sequence (pckA gene) coding forthe enzyme phosphoenolpyruvate carboxykinase (PEP carboxykinase) (EC4.1.1.49) is attenuated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: pMAK705ΔpckA (=pMAK705deltapckA)

FIG. 2: pMAK705ΔyjfA (=pMAK705deltayjfA)

FIG. 3: pMW218gdhA

FIG. 4: pMW219rhtC

FIG. 5: pMAK705Δ90bp (=pMAK705delta90bp)

The length data are to be understood as approxroximate data. Theabbreviations and designations used have the following meaning:

-   -   cat: Chloramphenicol resistance gene    -   rep-ts: Temperature-sensitive replication region of the plasmid        pSC101    -   pck1: Part of the 5′ region of the pckA gene    -   pck2: Part of the 3′ region of the pckA gene    -   ytfP′-yjfA′: DNA sequence containing truncated coding regions of        ytfP and yjfA    -   kan: Kanamycin resistance gene    -   gdhA: Glutamate dehydrogenase gene    -   rhtC: Threonine resistance-imparting gene

The abbreviations for the restriction enzymes have the following meaning

-   -   BamHI: restriction endonuclease from Bacillus amyloliquefaciens    -   BglIII: restriction endonuclease from Bacillus globigii    -   ClaI: restriction endonuclease from Caryphanon latum    -   EcoRI: restriction endonuclease from Escherichia coli    -   EcoRV: restriction endonuclease from Escherichia coli    -   HindIII: restriction endonuclease from Haemophilus influenzae    -   KpnI: restriction endonuclease from Klebsiella pneumoniae    -   PstI: restriction endonuclease from Providencia stuartii    -   PvuI: restriction endonuclease from Proteus vulgaris    -   SacI: restriction endonuclease from Streptomyces achromogenes    -   SalI: restriction endonuclease from Streptomyces albus    -   SmaI: restriction endonuclease from Serratia marcescens    -   XbaI: restriction endonuclease from Xanthomonas badrii    -   XhoI: restriction endonuclease from Xanthomonas holcicola

DETAILED DESCRIPTION OF THE INVENTION

Where L-amino acids or amino acids are mentioned in the following, thismeans one or more amino acids, including their salts, chosen from thegroup consisting of L-asparagine, L-threonine, L-serine, L-glutamate,L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine,L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine,L-tryptophan, L-homoserine and L-arginine. L-Threonine is particularlypreferred.

In this context the term “attenuation” describes the reduction orswitching-off, in a microorganism, of the intracellular activity of oneor more enzymes (proteins) which are coded for by the appropriate DNA,for example by using a weak promoter or a gene or allele which codes foran appropriate enzyme with low activity, or inactivating the appropriateenzyme (protein), and optionally combining these measures.

By attenuation measures, the activity or concentration of thecorresponding protein is in general reduced to 0 to 75%, 0 to 50%, 0 to25%, 0 to 10% or 0 to 5% of the activity or concentration of thewild-type protein or of the activity or concentration of the protein inthe starting microorganism.

The process is characterized in that the following steps are carriedout:

-   -   a) fermentation of microorganisms of the family        Enterobacteriaceae in which at least the pckA gene is        attenuated,    -   b) enrichment of the appropriate L-amino acid in the medium or        in the cells of the microorganisms of the family        Enterobacteriaceae, and    -   c) isolation of the desired L-amino acid.

The microorganisms provided by the present invention can produce L-aminoacids from glucose, sucrose, lactose, fructose, maltose, molasses,optionally starch or optionally cellulose, or from glycerol and ethanol.Said microorganisms are representatives of the family Enterobacteriaceaeselected from the genera Escherichia, Erwinia, Providencia and Serratia.The genera Escherichia and Serratia are preferred. The speciesEscherichia coli and Serratia marcescens may be mentioned in particularamong the genera Escherichia and Serratia respectively.

Examples of suitable strains, particularly L-threonine-producingstrains, of the genus Escherichia, especially of the species Escherichiacoli, are:

-   -   Escherichia coli TF427    -   Escherichia coli H4578    -   Escherichia coli KY10935    -   Escherichia coli VNIIgenetika MG442    -   Escherichia coli VNIIgenetika M1    -   Escherichia coli VNIIgenetika 472T23    -   Escherichia coli BKIIM B-3996    -   Escherichia coli kat 13    -   Escherichia coli KCCM-10132.

Examples of suitable L-threonine-producing strains of the genusSerratia, especially of the species Serratia marcescens, are:

-   -   Serratia marcescens HNr21    -   Serratia marcescens TLr156    -   Serratia marcescens T2000.

L-Threonine-producing strains of the family Enterobacteriaceaepreferably possess, inter alia, one or more genetic or phenotypiccharacteristics selected from the group comprising resistance toα-amino-β-hydroxyvaleric acid, resistance to thialysine, resistance toethionine, resistance to α-methylserine, resistance to diaminosuccinicacid, resistance to α-aminobutyric acid, resistance to borrelidine,resistance to rifampicin, resistance to valine analogues such as valinehydroxamate, resistance to purine analogues such as6-dimethylaminopurine, need for L-methionine, optionally partial andcompensable need for L-isoleucine, need for meso-diaminopimelic acid,auxotrophy in respect of threonine-containing dipeptides, resistance toL-threonine, resistance to L-homoserine, resistance to L-lysine,resistance to L-methionine, resistance to L-glutamic acid, resistance toL-aspartate, resistance to L-leucine, resistance to L-phenylalanine,resistance to L-serine, resistance to L-cysteine, resistance toL-valine, sensitivity to fluoropyruvate, defective threoninedehydrogenase, optionally capability for sucrose utilization,amplification of the threonine operon, amplification of homoserinedehydrogenase I-aspartate kinase I, preferably of the feedback-resistantform, amplification of homoserine kinase, amplification of threoninesynthase, amplification of aspartate kinase, optionally of thefeedback-resistant form, amplification of aspartate semialdehydedehydrogenase, amplification of phosphoenolpyruvate carboxylase,optionally of the feedback-resistant form, amplification ofphosphoenolpyruvate synthase, amplification of transhydrogenase,amplification of the RhtB gene product, amplification of the RhtC geneproduct, amplification of the YfiK gene product, amplification of malatequinone oxidoreductase and amplification of a pyruvate carboxylase andattenuation of acetic acid formation.

It has been found that the production of L-amino acids, especiallyL-threonine, by microorganisms of the family Enterobacteriaceae isimproved after attenuation and, in particular, switching-off of the pckAgene coding for PEP carboxykinase (EC 4.1.1.49).

The nucleotide sequence of the pcka gene of Escherichia coli has beenpublished by Medina et al. (Journal of Bacteriology 172, 7151-7156(1990)) and can also be taken from the genome sequence of Escherichiacoli published by Blattner et al. (Science 277, 1453-1462 (1997)). Thenucleotide sequence of the pckA gene of Escherichia coli is representedin SEQ ID No. 1 and the amino acid sequence of the corresponding geneproduct is represented in SEQ ID No. 2.

The pckA genes described in the above literature references can be usedaccording to the invention. It is also possible to use alleles of thepckA gene which result from the degeneracy of the genetic code or fromneutral sense mutations.

Attenuation can be achieved for example by reducing or switching off theexpression of the pcka gene or the catalytic properties of the enzymeprotein. Both measures may optionally be combined.

Gene expression can be reduced by an appropriate culture technique, bygenetic modification (mutation) of the signal structures of geneexpression, or by means of antisense RNA technology. Examples of signalstructures of gene expression are repressor genes, activator genes,operators, promoters, attenuators, ribosome binding sites, the startcodon and terminators. Those skilled in the art will find relevantinformation inter alia in e.g. Jensen and Hammer (Biotechnology andBioengineering 58, 191-195 (1998)), Carrier and Keasling (BiotechnologyProgress 15, 58-64 (1999)), Franch and Gerdes (Current Opinion inMicrobiology 3, 159-164 (2000)) and well-known textbooks on genetics andmolecular biology, for example the textbook by Knippers (“MolekulareGenetik” (“Molecular Genetics”), 6th edition, Georg Thieme Verlag,Stuttgart, Germany, 1995) or the textbook by Winnacker (“Gene und Klone”(“From Genes to Clones”), VCH Verlagsgesellschaft, Weinheim, Germany,1990).

Mutations which cause a change or reduction in the catalytic propertiesof enzyme proteins are known from the state of the art. Examples whichmay be mentioned are the studies of Qiu and Goodman (Journal ofBiological Chemistry 272, 8611-8617 (1997)), Yano et al. (Proceedings ofthe National Academy of Sciences USA 95, 5511-5515 (1998)) and Wente andSchachmann (Journal of Biological Chemistry 266, 20833-20839 (1991)).Surveys can be found in well-known textbooks on genetics and molecularbiology, e.g. the textbook by Hagemann (“Allgemeine Genetik” (“GeneralGenetics”), Gustav Fischer Verlag, Stuttgart, 1986).

Mutations to be taken into consideration are transitions, transversions,insertions and deletions. Depending on the effect of amino acid exchangeon the enzyme activity, the term missense mutations or nonsensemutations is used. Insertions or deletions of at least one base pair ina gene cause frame shift mutations, the result of which is that falseamino acids are incorporated or translation is terminated prematurely.Deletions of several codons typically lead to a complete loss of enzymeactivity. Instructions for the production of such mutations form paft ofthe state of the art and can be found in well-known textbooks ongenetics and molecular biology, e.g. the textbook by Knippers(“Molekulare Genetik” (“Molecular Genetics”), 6th edition, Georg ThiemeVerlag, Stuttgart, Germany, 1995), the textbook by Winnacker (“Gene undKlone” (“From Genes to Clones”), VCH Verlagsgesellschaft, Weinheim,Germany, 1990) or the textbook by Hagemann (“Allgemeine Genetik”(“General Genetics”), Gustav Fischer Verlag, Stuttgart, 1986).

An example of a plasmid by means of which the pckA gene of Escherichiacoli can be attenuated and, in particular, switched off byposition-specific mutagenesis is plasmid pMAK705ΔpckA (FIG. 1). Itcontains only part of the 5′ region and part of the 3′ region of thepckA gene. A 349 bp segment of the coding region is missing (deletion).The sequence of this DNA, which can be used for mutagenesis of the pckAgene, is represented in SEQ ID No. 3.

The deletion mutation of the pckA gene can be incorporated into suitablestrains by gene or allele exchange.

A common method is the method of gene exchange using a conditionallyreplicating pSC101 derivative, pMAK705, as described by Hamilton et al.(Journal of Bacteriology 174, 4617-4622 (1989)). Other methods describedin the state of the art, for example that of Martinez-Morales et al.(Journal of Bacteriology, 7143-7148 (1999)) or that of Boyd et al.(Journal of Bacteriology 182, 842-847 (2000)), can also be used.

When exchange has been carried out, the form of the ΔpckA allelerepresented in SEQ ID No. 4, which is a further subject of theinvention, is present in the strain in question.

Mutations in the pckA gene or mutations involving expression of the pckAgene can also be transferred to different strains by conjugation ortransduction.

Furthermore, for the production of L-amino acids, especiallyL-threonine, with strains of the family Enterobacteriaceae, it can beadvantageous not only to attenuate the pckA gene but also to amplify oneor more enzymes of the known threonine biosynthetic pathway, or enzymesof the anaplerotic metabolism, or enzymes for the production of reducednicotinamide adenine dinucleotide phosphate.

In this context the term “amplification” describes the increase in theintracellular activity, in a microorganism, of one or more enzymes orproteins which are coded for by the appropriate DNA, for example byincreasing the copy number of the gene(s), using a strong promoter orusing a gene coding for an appropriate enzyme or protein with a highactivity, and optionally combining these measures.

By amplification measures, in particular over-expression, the activityor concentration of the corresponding protein is in general increased byat least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up toa maximum of 1000% or 2000%, based on that of the wild-type protein orthe activity or concentration of the protein in the startingmicroorganism.

Thus, for example, one or more genes selected from the group comprising:

-   -   the thrABC operon coding for aspartate kinase, homoserine        dehydrogenase, homoserine kinase and threonine synthase (U.S.        Pat. No. 4,278,765),    -   the pyc gene coding for pyruvate carboxylase DE-A-19 831 609),    -   the pps gene coding for phosphoenolpyruvate synthase (Molecular        and General Genetics 231, 332 (1992)),    -   the ppc gene coding for phosphoenolpyruvate carboxylase (Gene        31, 279-283 (1984)),    -   the pntA and pntB genes coding for transhydrogenase (European        Journal of Biochemistry 158, 647-653 (1986)),    -   the rhtB gene for homoserine resistance (EP-A-0994190), and    -   the rhtC gene for threonine resistance (EP-A-1013765),    -   the gdhA gene coding for glutamate dehydrogenase (Gene        27:193-199 (1984)        can be simultaneously amplified and, in particular,        overexpressed.

Furthermore, for the production of L-amino acids, especiallyL-threonine, it can be advantageous not only to attenuate the pckA genebut also to attenuate and, in particular, switch off one or more genesselected from the group comprising:

-   -   the tdh gene coding for threonine dehydrogenase (Ravnikar and        Somerville, Journal of Bacteriology 169, 4716-4721 (1987)),    -   the mdh gene coding for malate dehydrogenase (EC 1.1.1.37)        (Vogel et al., Archives in Microbiology 149, 36-42 (1987)),    -   the gene product of the open reading frame (orf) yjfA (Accession        Number AAC77180 of the National Center for Biotechnology        Information (NCBI, Bethesda, Md., USA) and SEQ ID No. 5), and    -   the gene product of the open reading frame (orf) ytfP (Accession        Number AAC77179 of the National Center for Biotechnology        Information (NCBI, Bethesda, Md., USA) and SEQ ID No. 5),        or to reduce the expression.

It is preferred to attenuate the open reading frame yjfA and/or the openreading frame ytfP.

It is also possible according to the invention to attenuate the openreading frames yjfA and/or ytfp independently of the pckA gene, in orderto achieve an improvement in the amino acids, in particular L-threonineproduction.

The invention accordingly also provides a process, characterized in thatthe following steps are carried out:

-   -   d) fermentation of microorganisms of the Enterobacteriaceae        family in which at least the open reading frame yjfA and/or ytfP        is attenuated,    -   e) enrichment of the L-amino acid in the medium or in the cells        of the microorganisms of the Enterobacteriaceae family, and    -   f) isolation of the L-threonine, constituents of the        fermentation broth and the biomass in its entirety or portions        thereof optionally being isolated as a solid product together        with the L-amino acid.

An example of a plasmid by means of which the open reading frames yjfAand ytfP of Escherichia coli can be attenuated and, in particular,switched off by position-specific mutagenesis is plasmid pMAK705ΔyjfA(FIG. 2). It contains only the 5′ and 3′ flanks of the ytfP-yjfA region,including very short residues of the open reading frames yjfA and ytfp.A 337 bp long part of the ytfP-yjfA region is missing (deletion). Thesequence of this DNA, which can be used for mutagenesis of the ytfP-yjfAregion, is represented in SEQ ID No. 6.

An further example of a plasmid by means of which the open readingframes yjfA and ytfP of Escherichia coli can be attenuated and, inparticular, switched off by position-specific mutagenesis is the plasmidpMAK705Δ90bp (FIG. 5). It also contains only the 5′ and 31 flanks of theytfP-yjfA region including very short residues of the open readingframes yjfA and ytfp. A 90 bp long part of the ytfP-yjfA region ismissing (deletion). The sequence of this DNA, which can be used formutagenesis of the ytfP-yjfA region, is represented in SEQ ID No. 7.

This deletion mutation can be incorporated into suitable strains by geneor allele replacement. It is also possible to transfer mutations in theopen reading frames yjfA and/or ytfP or mutations affecting expressionof these open reading frames into various strains by conjugation ortransduction.

When replacement has been carried out, the form of the ΔytfP and ΔyjfAallele represented in SEQ ID No. 6 or SEQ ID No. 7, which are a furthersubject of the invention, is present in the strain in question.

Furthermore, for the production of L-amino acids, especiallyL-threonine, it can be advantageous, in addition to the individual orjoint attenuation of the pckA gene or of the open reading frames yjfAand/or ytfP, to switch off undesired secondary reactions (Nakayama:“Breeding of Amino Acid Producing Microorganisms”, in: Overproduction ofMicrobial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press,London, UK, 1982).

The microorganisms prepared according to the invention can be cultivatedby the batch process or the fed batch process. A summary of knowncultivation methods is provided in the textbook by Chmiel(Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (BioprocessTechnology 1. Introduction to Bioengineering) (Gustav Fischer Verlag,Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren undperiphere Einrichtungen (Bioreactors and Peripheral Equipment) (ViewegVerlag, Brunswick/Wiesbaden, 1994)).

The culture medium to be used must appropriately meet the demands of theparticular strains. Descriptions of culture media for variousmicroorganisms can be found in the handbook “Manual of Methods forGeneral Bacteriology” of the American Society for Bacteriology(Washington D.C., USA, 1981).

Carbon sources which can be used are sugars and carbohydrates, e.g.glucose, sucrose, lactose, fructose, maltose, molasses, starch andoptionally cellulose, oils and fats, e.g. soya oil, sunflower oil,groundnut oil and coconut fat, fatty acids, e.g. palmitic acid, stearicacid and linoleic acid, alcohols, e.g. glycerol and ethanol, and organicacids, e.g. acetic acid. These substances can be used individually or asa mixture.

Nitrogen sources which can be used are organic nitrogen-containingcompounds such as peptones, yeast extract, meat extract, malt extract,corn steep liquor, soya bean flour and urea, or inorganic compounds suchas ammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate and ammonium nitrate. The nitrogen sources can be usedindividually or as a mixture.

Phosphorus sources which can be used are phosphoric acid, potassiumdihydrogenphosphate or dipotassium hydrogenphosphate or thecorresponding sodium salts. The culture medium must also contain metalsalts, e.g. magnesium sulfate or iron sulfate, which are necessary forgrowth. Finally, essential growth-promoting substances such as aminoacids and vitamins can be used in addition to the substances mentionedabove. Suitable precursors can also be added to the culture medium. Saidfeed materials can be added to the culture all at once or fed inappropriately during cultivation.

The pH of the culture is controlled by the appropriate use of basiccompounds such as sodium hydroxide, potassium hydroxide, ammonia oraqueous ammonia, or acid compounds such as phosphoric acid or sulfuricacid. Foaming can be controlled using antifoams such as fatty acidpolyglycol esters. The stability of plasmids can be maintained by addingsuitable selectively acting substances, e.g. antibiotics, to the medium.Aerobic conditions are maintained by introducing oxygen oroxygen-containing gaseous mixtures, e.g. air, into the culture. Thetemperature of the culture is normally 25° C. to 45° C. and preferably30° C. to 40° C. The culture is continued until the formation of L-aminoacids or L-threonine has reached a maximum. This objective is normallyachieved within 10 hours to 160 hours.

L-Amino acids can be analyzed by means of anion exchange chromatographyfollowed by ninhydrin derivation, as described by Spackman et al.(Analytical Chemistry 30, 1190 (1958)), or by reversed phase HPLC, asdescribed by Lindroth et al. (Analytical Chemistry 51, 1167-1174(1979)).

A pure culture of the Escherichia coli K-12 strain DH5α/pMAK705 wasdeposited on 8^(th) Sep. 2000 at the Deutsche Sammlung fürMikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1B, D-3300Braunschweig, Germany (DSMZ=German Collection of Microorganisms and CellCultures, Braunschweig, Germany) in accordance with the Budapest Treatyas DSM 13720.

A pure culture of the Escherichia coli K-12 strain MG442ΔpckA wasdeposited on 2^(nd) Oct. 2000 at the Deutsche Sammlung fürMikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1B, D-3300Braunschweig, Germany (DSMZ=German Collection of Microorganisms and CellCultures, Braunschweig, Germany) in accordance with the Budapest Treatyas DSM 13761.

A pure culture of the Escherichia coli K-12 strainB-3996kurΔtdhΔpckA/pVIC40 was deposited on 9^(th) Mar. 2001 at theDeutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (DSM),Mascheroder Weg 1B, D-3300 Braunschweig, Germany (DSMZ=German Collectionof Microorganisms and Cell Cultures, Braunschweig, Germany) inaccordance with the Budapest Treaty as DSM 14150.

A pure culture of the Escherichia coli K-12 strain MG442Δ90yjfA wasdeposited on 9^(th) May 2001 at the Deutsche Sammlung fürMikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1B, D-3300Braunschweig, Germany (DSMZ=German Collection of Microorganisms and CellCultures, Braunschweig, Germany) in accordance with the Budapest Treatyas DSM 14289.

It is also possible according to the invention individually to attenuatethe open reading frames ytfP and yjfA in order to improve the productionof L-amino acids.

The process according to the invention is used for the preparation ofL-amino acids, e.g. L-threonine, L-isoleucine, L-methionine,L-homoserine and L-lysine, especially L-threonine, by fermentation.

The present invention is illustrated in greater detail below with theaid of Examples.

The isolation of plasmid DNA from Escherichia coli and all thetechniques for restriction, Klenow treatment and alkaline phosphatasetreatment were carried out as described by Sambrook et al. (Molecularcloning—A laboratory manual (1989), Cold Spring Harbor LaboratoryPress). Unless indicated otherwise, the transformation of Escherichiacoli was carried out as described by Chung et al. (Proceedings of theNational Academy of Sciences USA 86, 2172-2175 (1989)).

The incubation temperature for the preparation of strains andtransformants was 37° C. Temperatures of 30° C. and 44° C. were used inthe gene exchange process of Hamilton et al.

EXAMPLE 1

Construction of the Deletion Mutation of the pckA Gene

Parts of the 5′ and 3′ regions of the pckA gene of Escherichia coli K12were amplified using the polymerase chain reaction (PCR) and syntheticoligonucleotides. The nucleotide sequence of the pckA gene in E. coliK12 MG1655 (SEQ ID No. 1) was used to synthesize the following PCRprimers (MWG Biotech, Ebersberg, Germany):

pckA′5′-1: 5′ - GATCCGAGCCTGACAGGTTA - 3′ (SEQ ID NO:8) pckA′5′-2: 5′ -GCATGCGCTCGGTCAGGTTA - 3′ (SEQ ID NO:9) pckA′3′-1: 5′ -AGGCCTGAAGATGGCACTATCG - 3′ (SEQ ID NO:10) pckA′3′-2: 5′ -CCGGAGAAGCGTAGGTGTTA - 3′. (SEQ ID NO:11)The chromosomal E. coli K12 MG1655 DNA used for the PCR was isolatedwith “Qiagen Genomic-tips 100/G” (QIAGEN, Hilden, Germany) according tothe manufacturer's instructions. An approx. 500 bp DNA fragment from the5′ region of the pckA gene (denoted as pck1) and an approx. 600 bp DNAfragment from the 3′ region of the pckA gene (denoted as pck2) could beamplified with the specific primers under standard PCR conditions (Inniset al. (1990), PCR Protocols. A Guide to Methods and Applications,Academic Press) using Taq DNA polymerase (Gibco-BRL, Eggenstein,Germany). The PCR products were each ligated with vector pCR2.1TOPO(TOPO TA Cloning Kit, Invitrogen, Groningen, The Netherlands) accordingto the manufacturer's instructions and transformed into E. coli strainTOP10F′. Plasmid-carrying cells were selected on LB agar containing 50μg/ml of ampicillin. After isolation of the plasmid DNA, vectorpCR2.1TOPOpck2 was cleaved with the restriction enzymes StuI and XbaIand, after separation in 0.8% agarose gel, the pck2 fragment wasisolated with the aid of the QIAquick Gel Extraction Kit (QIAGEN,Hilden, Germany). After isolation of the plasmid DNA, vectorpCR2.1TOPOpck1 was cleaved with the enzymes EcoRV and XbaI and ligatedto the isolated pck2 fragment. The E. coli strain DH5α was transformedwith the ligation mixture and plasmid-carrying cells were selected on LBagar containing 50 μg/ml of ampicillin. After isolation of the plasmidDNA, control cleavage with the enzymes SpeI and XbaI was used to detectplasmids containing, in cloned form, the mutagenic DNA sequencerepresented in SEQ ID No. 3. One of the plasmids was denoted aspCR2.1TOPOΔpckA.

EXAMPLE 2

Construction of Exchange Vector pMAK705ΔpckA

After restriction with the enzymes SpeI and XbaI and separation in 0.8%agarose gel, the pckA allele described in Example 1 was isolated fromvector pCR2.1TOPOΔpckA and ligated to plasmid pMAK705 (Hamilton et al.,Journal of Bacteriology 174, 4617-4622 (1989)) which had been digestedwith the enzyme XbaI. DH5α was transformed with the ligation mixture andplasmid-carrying cells were selected on LB agar containing 20 μg/ml ofchloramphenicol. After isolation of the plasmid DNA and cleavage withthe enzymes HpaI, KpnI, HindIII, SalI and PstI, successful cloning wasdetected. The exchange vector formed, pMAK705ΔpckA (=pMAK705deltapckA),is shown in FIG. 1.

EXAMPLE 3

Position-specific Mutagenesis of the pckA Gene in the E. coli StrainMG442

The L-threonine-producing E. coli strain MG442 is described in patentU.S. Pat. No. 4,278,765 and deposited in the Russian National Collectionof Industrial Microorganisms (VKPM, Moscow, Russia) as CMIM B-1628.

The strain MG442 has a resistance to α-amino-β-hydroxyvaleric acid andhas an optionally partial and compensable need for L-isoleucine.

For exchange of the chromosomal pckA gene for the plasmid-coded deletionconstruct, MG442 was transformed with plasmid pMAK705ΔpckA. The geneexchange was carried out by the selection method described by Hamiltonet al. (Journal of Bacteriology 174, 4617-4622 (1989)) and was verifiedby standard PCR methods (Innis et al. (1990), PCR Protocols. A Guide toMethods and Applications, Academic Press) using the followingoligonucleotide primers:

pckA′5′-1: 5′ - GATCCGAGCCTGACAGGTTA - 3′ (SEQ ID NO:8) pckA′3′-2: 5′ -CCGGAGAAGCGTAGGTGTTA - 3′ (SEQ ID NO:11)The strain obtained was denoted as MG442ΔpckA.

EXAMPLE 4

Preparation of L-threonine with the Strain MG442ΔpckA

MG442ΔpckA was cultivated on minimum medium of the followingcomposition: 3.5 g/l of Na₂HPO₄.2H₂O, 1.5 g/l of KH₂PO₄, 1 g/l of NH₄Cl,0.1 g/l of MgSO₄.7H₂O, 2 g/l of glucose and 20 g/l of agar. Theformation of L-threonine was checked in 10 ml batch cultures containedin 100 ml Erlenmeyer flasks. These were inoculated with 10 ml of apreculture medium of the following composition: 2 g/l of yeast extract,10 g/l of (NH₄)₂SO₄, 1 g/l of KH₂PO₄, 0.5 g/l of MgSO₄ _(.)7H₂O, 15 g/lof CaCO₃ and 20 g/l of glucose, and incubated for 16 hours at 37° C. and180 rpm on an ESR incubator from Kühner AG (Birsfelden, Switzerland).250 μl of this preculture were transferred to 10 ml of a productionmedium (25 g/l of (NH₄)₂SO₄, 2 g/l of KH₂PO₄, 1 g/l of MgSO₄.7H₂O, 0.03g/l of FeSO₄.7H₂O, 0.018 g/l of MnSO₄.1H₂O, 30 g/l of CaCO₃, 20 g/l ofglucose) and incubated for 48 hours at 37° C. After incubation, theoptical density (OD) of the culture suspension was determined with anLP2W photometer from Dr. Lange (Berlin, Germany) at a measurementwavelength of 660 nm.

The concentration of L-threonine formed was then determined in thesterile-filtered culture supernatant with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by means of ion exchangechromatography and postcolumn reaction with ninhydrin detection.

The result of the experiment is shown in Table 1.

TABLE 1 OD L-Threonine Strain (660 nm) g/l MG442 6.0 1.5 MG442ΔpckA 5.43.7

EXAMPLE 5

Preparation of L-threonine with the Strain MG442ΔpckA/pMW218gdhA

5.1 Amplification and Cloning of the gdhA Gene

The glutamate dehydrogenase gene from Escherichia coli K12 is amplifiedusing the polymerase chain reaction (PCR) and syntheticoligonucleotides. Starting from the nucleotide sequence for the gdhAgene in E. coli K12 MG1655 (gene library: Accession No. AE000270 and No.AE000271) PCR primers are synthesized (MWG Biotech, Ebersberg, Germany):

Gdh1: 5′ - TGAACACTTCTGGCGGTACG - 3′ (SEQ ID NO:12) Gdh2: 5′ -CCTCGGCGAAGCTAATATGG - 3′ (SEQ ID NO:13)The chromosomal E. coli K12 MG1655 DNA employed for the PCR is isolatedaccording to the manufacturers instructions with “QIAGEN Genomic-tips100/G” (QIAGEN, Hilden, Germany). A DNA fragment approx. 2150 bp insize, which comprises the gdhA coding region and approx. 350 bp5′-flanking and approx. 450 bp 3′-flanking sequences, can be amplifiedwith the specific primers under standard PCR conditions (Innis et al.:PCR Protocols. A Guide to Methods and Applications, 1990, AcademicPress) with the Pfu-DNA polymerase (Promega Corporation, Madison, USA).The PCR product is cloned in the plasmid pCR2.1TOPO and transformed inthe E. coli strain TOP10 (Invitrogen, Leek, The Netherlands, ProductDescription TOPO TA Cloning Kit, Cat. No. K4500-01). Successful cloningis demonstrated by cleavage of the plasmid pCR2.1TOPOgdhA with therestriction enzymes EcoRI and EcoRV. For this, the plasmid DNA isisolated by means of the “QIAprep Spin Plasmid Kits” (QIAGEN, Hilden,Germany) and, after cleavage, separated in a 0.8% agarose gel.5.2 Cloning of the gdhA Gene in the Plasmid Vector pMW218

The plasmid pCR2.1TOPOgdhA is cleaved with the enzyme EcoRI, thecleavage batch is separated on 0.8% agarose gel and the gdhA fragment2.1 kbp in size is isolated with the aid of the “QIAquick Gel ExtractionKit” (QIAGEN, Hilden, Germany). The plasmid pMW218 (Nippon Gene, Toyama,Japan) is cleaved with the enzyme EcoRI and ligated with the gdhAfragment. The E. coli strain DH5α is transformed with the ligation batchand pMW218-carrying cells are selected by plating out on LB agar(Lennox, Virology 1955, 1: 190), to which 20 μg/ml kanamycin are added.

Successful cloning of the gdhA gene can be demonstrated after plasmidDNA isolation and control cleavage with EcoRI and EcoRV. The plasmid iscalled pMW218gdhA (FIG. 3).

5.3 Preparation of the Strain MG442ΔpckA/pMW218gdhA

The strain MG442ΔpckA obtained in Example 3 and the strain MG442 aretransformed with the plasmid pMW218gdhA and transformants are selectedon LB agar, which is supplemented with 20 μg/ml kanamycin. The strainsMG442ΔpckA/pMW218gdhA and MG442/pMW218gdhA are formed in this manner.

5.4 Preparation of L-threonine

The preparation of L-threonine by the strains MG442ΔpckA/pMW218gdhA andMG442/pMW218gdhA is tested as described in Example 4. The minimal mediumand the preculture medium are additionally supplemented with 20 μg/mlkanamycin.

The result of the experiment is summarized in Table 2.

TABLE 2 OD L-Threonine Strain (660 nm) g/l MG442 6.0 1.5 MG442ΔpckA 5.43.7 MG442/pMW218gdhA 5.6 2.6 MG442ΔpckA/pMW218gdhA 5.5 4.0

EXAMPLE 6

Preparation of L-threonine with the Strain MG442ΔpckA/pMW219rhtC

6.1 Amplification of the rhtC Gene

The rhtC gene from Escherichia coli K12 is amplified using thepolymerase chain reaction (PCR) and synthetic oligonucleotides. Startingfrom the nucleotide sequence for the rhtC gene in E. coli K12 MG1655(gene library: Accession No. AE000458, Zakataeva et al. (FEBS Letters452, 228-232 (1999)), PCR primers are synthesized (MWG Biotech,Ebersberg, Germany):

RhtC1: 5′ - CTGTTAGCATCGGCGAGGCA - 3′ (SEQ ID NO:14) RhtC2: 5′ -GCATGTTGATGGCGATGACG - 3′ (SEQ ID NO:15)The chromosomal E. coli K12 MG1655 DNA employed for the PCR is isolatedaccording to the manufacturers instructions with “QIAGEN Genomic-tips100/G” (QIAGEN, Hilden, Germany). A DNA fragment approx. 800 bp in sizecan be amplified with the specific primers under standard PCR conditions(Innis et al.: PCR Protocols. A Guide to Methods and Applications, 1990,Academic Press) with Pfu-DNA polymerase (Promega Corporation, Madison,USA).6.2 Cloning of the rhtC Gene in the Plasmid Vector pMW219

The plasmid pMW219 (Nippon Gene, Toyama, Japan) is cleaved with theenzyme SamI and ligated with the rhtC-PCR fragment. The E. coli strainDH5α is transformed with the ligation batch and pMW219-carrying cellsare selected on LB agar, which is supplemented with 20 μg/ml kanamycin.Successful cloning can be demonstrated after plasmid DNA isolation andcontrol cleavage with KpnI, HindIII and NcoI. The plasmid pMW219rhtC isshown in FIG. 4.

6.3 Preparation of the Strain MG442ΔpckA/pMW219rhtC

The strain MG442ΔpckA obtained in Example 3 and the strain MG442 aretransformed with the plasmid pMW219rhtC and transformants are selectedon LB agar, which is supplemented with 20 μg/ml kanamycin. The strainsMG442ΔpckA/pMW219rhtC and MG442/pMW219rhtC are formed in this manner.

6.4 Preparation of L-threonine

The preparation of L-threonine by the strains MG442ΔpckA/pMW219rhtC andMG442/pMW219rhtC is tested as described in Example 4. The minimal mediumand the preculture medium are additionally supplemented with 20 μg/mlkanamycin. The result of the experiment is summarized in Table 3.

TABLE 3 OD L-Threonine Strain (660 nm) g/l MG442 6.0 1.5 MG442ΔpckA 5.43.7 MG442/pMW219rhtC 5.2 2.9 MG442ΔpckA/pMW219rhtC 4.8 4.4

EXAMPLE 7

Preparation of L-threonine with the Strain B-3996kurΔtdhΔpckA/pVIC40

The L-threonine-producing E. coli strain B-3996 is described in U.S.Pat. No. 5,175,107 and deposited at the Russian National Collection forIndustrial Microorganisms (VKPM, Moscow, Russia).

The strain B-3996 has, inter alia, a resistance toα-amino-β-hydroxyvaleric acid, has an attenuated, in particularswitched-off, or defective threonine dehydrogenase, has an enhancedhomoserine dehydrogenase I aspartate kinase I in the feed back resistantform, has an optionally partial and compensable need for L-isoleucineand has the ability to utilize sucrose.

7.1 Preparation of the Strain B-3996kurΔtdhΔpckA/pVIC40

After culture in antibiotic-free complete medium for approximately tengenerations, a derivative of strain B-3996 which no longer contains theplasmid pVIC40 is isolated. The strain formed is streptomycin-sensitiveand is designated B-3996kur.

The method described by Hamilton et al. (Journal of Bacteriology (1989)171: 4617-4622), which is based on the use of the plasmid pMAK705 with atemperature-sensitive replicon, was used for incorporation of a deletioninto the tdh gene. The plasmid pDR121 (Ravnikar and Somerville, Journalof Bacteriology (1987) 169:4716-4721) contains a DNA fragment from E.coli 3.7 kilo-base pairs (kbp) in size, on which the tdh gene is coded.To generate a deletion of the tdh gene region, pDR121 is cleaved withthe restriction enzymes ClaI and EcoRV and the DNA fragment 5 kbp insize isolated is ligated, after treatment with Klenow enzyme. Theligation batch is transformed in the E. coli strain DH5α andplasmid-carrying cells are selected on LB agar, to which 50 μg/mlampicillin are added.

Successful deletion of the tdh gene can be demonstrated after plasmidDNA isolation and control cleavage with EcoRI. The EcoRI fragment 1.7kbp in size is isolated, and ligated with the plasmid pMAK705, which ispartly digested with EcoRI. The ligation batch is transformed in DH5αand plasmid-carrying cells are selected on LB agar, to which 20 μg/mlchloramphenicol are added. Successful cloning is demonstrated afterisolation of the plasmid DNA and cleavage with EcoRI. The pMAK705derivative formed is designated pDM32.

For the gene replacement, B-3996kur is transformed with the plasmidpDM32. The replacement of the chromosomal tdh gene with theplasmid-coded deletion construct is carried out by the selection processdescribed by Hamilton et al. and is verified by standard PCR methods(Innis et al. (1990), PCR Protocols. A Guide to Methods andApplications, Academic Press) with the following oligonucleotideprimers:

Tdh1: 5′-TCGCGACCTATAAGTTTGGG-3′ (SEQ ID NO:16) Tdh2:5′-AATACCAGCCCTTGTTCGTG-3′. (SEQ ID NO:17)The strain formed is tested for kanamycin sensitivity and is designatedB-3996kurΔtdh.

For the position-specific mutagenesis of the pckA gene, B-3996kurΔtdh istransformed with the replacement vector pMAK705ΔpckA described inExample 2. The replacement of the chromosomal pckA gene by theplasmid-coded deletion construct is carried out as described in Example3. The strain obtained is called B-3996kurΔtdhΔpckA.

B-3996kurΔtdh and B-3996kurΔtdhΔpckA are transformed with the plasmidpVIC40 isolated from B-3996 and plasmid-carrying cells are selected onLB agar with 20 μg/ml streptomycin. In each case a selected individualcolony is called B-3996kurΔtdh/pVIC40 and B-3996kurΔtdhΔpckA/pVIC40.

7.2 Preparation of L-threonine

The preparation of L-threonine by the strains B-3996kurΔtdh/pVIC40 andB-3996kurΔtdhΔpckA/pVIC40 is tested as described in Example 4. Theminimal medium, the preculture medium and the production medium areadditionally supplemented with 20 μg/ml streptomycin.

The result of the experiment is summarized in Table 4.

TABLE 4 OD L-Threonine Strain (660 nm) g/l B-3996kurΔtdh/pVIC40 4.7 6.26B-3996kurΔtdhΔpckA/pVIC40 4.9 8.92

EXAMPLE 8

Preparation of L-lysine with the Strain TOC21RΔpckA

The L-lysine-producing E. coli strain pDA1/TOC21R is described in thepatent application F-A-2511032 and deposited at the Collection Nationalede Culture de Microorganisme (CNCM=National Microorganism CultureCollection, Pasteur Institute, Paris, France) under number I-167. Thestrain and the plasmid-free host are also described by Dauce-Le Reverendet al. (European Journal of Applied Microbiology and Biotechnology15:227-231 (1982)) under the name TOCR21/pDA1.

8.1 Position-specific Mutagenesis of the pckA Gene in the E. coli StrainTOC21R

After culture in antibiotic-free LB medium for approximately sixgenerations, a derivative of strain pDA1/TOC21R which no longer containsthe plasmid pDA1 is isolated. The strain formed istetracycline-sensitive and is called TOC21R.

For replacement of the chromosomal pckA gene by the plasmid-codeddeletion construct, TOC21R is transformed with the plasmid pMAK705ΔpckA(Example 2). The gene replacement is carried out by the selection methoddescribed by Hamilton et al. (1989) Journal of Bacteriology 174,4617-4622) and is verified by standard PCR methods (Innis et al. (1990)PCR Protocols. A Guide to Methods and Applications, Academic Press) withthe following oligonucleotide primers:

pckA′5′-1: 5′ - GATCCGAGCCTGACAGGTTA - 3′ (SEQ ID NO:8) pckA′3′-2: 5′ -CCGGAGAAGCGTAGGTGTTA - 3′ (SEQ ID NO:11)The strain obtained is called TOC21RΔpckA.8.2 Preparation of L-lysine with the Strain TOC21RΔpckA

The formation of L-lysine by the strains TOC21RΔ pckA and TOC21R ischecked in batch cultures of 10 ml contained in 100 ml conical flasks.For this, 10 ml of preculture medium of the following composition: 2 g/lyeast extract, 10 g/l (NH₄)₂SO₄, 1 g/l KH₂PO₄, 0.5 g/l MgSO₄*7H₂O, 15g/l CaCO₃, 20 g/l glucose are inoculated and the batch is incubated for16 hours at 37° C. and 180 rpm on an ESR incubator from Kühner AG(Birsfelden, Switzerland). 250 μl of this preculture are transinoculatedinto 10 ml of production medium (25 g/l (NH₄)₂SO₄, 2 g/l KH₂PO₄, 1 g/lMgSO₄*7H₂O, 0.03 g/l FeSO₄*7H₂O, 0.018 g/l MnSO₄*1H₂O, 30 g/l CaCO₃, 20g/l glucose, 25 mg/l L-isoleucine and 5 mg/l thiamine) and the batch isincubated for 72 hours at 37° C. After the incubation the opticaldensity (OD) of the culture suspension is determined with an LP2Wphotometer from Dr. Lange (Berlin, Germany) at a measurement wavelengthof 660 nm.

The concentration of L-lysine formed is then determined in thesterile-filtered culture supernatant with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column reaction with ninhydrin detection.

The result of the experiment is shown in Table 5.

TABLE 5 OD L-Lysine Strain (660 nm) g/l TOC21R 1.0 1.14 TOC21RΔpckA 1.01.27

EXAMPLE 9

Preparation of L-isoleucine with the StrainB-3996kurΔtdhilvA⁺ΔpckA/pVIC40

9.1 Preparation of the Strain B-3996kurΔtdhilvA⁺ΔpckA/pVIC40

The strain B-3996kurΔtdh, which is in need of L-isoleucin, obtained inExample 7.1 is transduced with the aid of the phage P1kc (Lennox,Virology 1, 190-206 (1955); Miller, Experiments in Molecular Genetics,Cold Spring Harbor Laboratory 1972) and L-isoleucine-prototrophictransductants are isolated.

For this, the phage P1kc is multiplied on the strain MG1655 (Guyer etal., Cold Spring Harbor Symposium of Quantitative Biology 45, 135-140(1981) and Blattner et al., Science 277, 1453-1462 (1997))and the phagelysate is employed for the transduction of the strain B-3996kurΔtdh. Themultiplicity of the infection is approximately 0.2. Selection forL-isoleucine-prototrophic transductants is carried out on minimal agar,which contains 2 g/l glucose and 10 mg/l L-threonine. AnL-isoleucine-prototrophic transductant is isolated, smeared on to LBagar for purification or isolation and called B-3996kurΔtdhilvA⁺.

The pckA gene of the strain B-3996kurΔtdhilvA⁺is then replaced, asdescribed in Example 3, by the ΔpckA allele prepared in Example 1 and 2.The strain obtained is called B-3996kurΔtdhilvA⁺ΔpckA.

The strains B-3996kurΔtdhilvA⁺and B-3996kurΔtdhilvA⁺ΔpckA aretransformed with the plasmid pVIC40 isolated from strain B-3996 andplasmid-carrying cells are selected on LB agar, which is supplementedwith 20 μg/ml streptomycin. In each case a selected individual colony iscalled B-3996kurΔtdhilvA⁺ΔpckA/pVIC40 and B-3996kurΔtdhilvA⁺/pVIC40.

9.2 Preparation of L-isoleucine

The preparation of L-isoleucine by the strains B-3996kurΔtdhilvA⁺/pVIC40and B-3996kurΔtdhilvA⁺ΔpckA/pVIC40 is tested under the test conditionsas described in Example 4. The minimal medium, the preculture medium andthe production medium are additionally supplemented with 20 μg/mlstreptomycin.

The result of the experiment is shown in Table 6.

TABLE 6 OD L-Isoleucine Strain (660 nm) mg/l B-3996kurΔtdhilvA⁺/pVIC405.8 57 B-3996kurΔtdhilvA⁺ΔpckA/pVIC40 5.7 70

EXAMPLE 10

Preparation of L-valine with the Strain B-12288ΔpckA

The L-valine-producing E. coli strain AJ 11502 is described in thepatent specification U.S. Pat. No. 4,391,907 and deposited at theNational Center for Agricultural Utilization Research (Peoria, Ill.,USA) as NRRL B-12288.

10.1 Position-specific Mutagenesis of the pckA Gene in the E. coliStrain B-1288

After culture in antibiotic-free LB medium for approximately sixgenerations, a plasmid-free derivative pf strain AJ 11502 is isolated.The strain formed is ampicillin-sensitive and is called AJ11502kur.

For replacement of the chromosomal pckA gene by the plasmid-codeddeletion construct, AJ11502kur is transformed with the plasmidpMAK705ΔpckA (see Example 2). The gene replacement is carried out by theselection method described by Hamilton et al. (1989) Journal ofBacteriology 174, 4617-4622) and is verified by standard PCR methods(Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications,Academic Press) with the following oligonucleotide primers:

pckA′5′-1: 5′ - GATCCGAGCCTGACAGGTTA - 3′ (SEQ ID NO:8) pckA′3′-2: 5′ -CCGGAGAAGCGTAGGTGTTA - 3′ (SEQ ID NO:11)The strain obtained is called AJ11502kurΔpckA. The plasmid described inthe patent specification U.S. Pat. No. 4,391,907, which carries thegenetic information in respect of valine production, is isolated fromstrain NRRL B-12288. The strain AJ11502kurΔpckA is transformed with thisplasmid. One of the transformants obtained is called B-12288ΔpckA.10.2 Preparation of L-valine with the Strain B-12288ΔpckA

The formation of L-valine by the strains B-12288ΔpckA and NRRL B-12288is checked in batch cultures of 10 ml contained in 100 ml conicalflasks. For this, 10 ml of preculture medium of the followingcomposition: 2 g/l yeast extract, 10 g/l (NH₄)₂SO₄, 1 g/l KH₂PO₄, 0.5g/l MgSO₄*7H₂O, 15 g/l CaCO₃, 20 g/l glucose and 50 mg/l ampicillin areinoculated and the batch is incubated for 16 hours at 37° C. and 180 rpmon an ESR incubator from Küthner AG (Birsfelden, Switzerland). 250 μl ofthis preculture are transinoculated into 10 ml of production medium (25g/l (NH₄)₂SO₄, 2 g/l KH₂PO₄, 1 g/l MgSO₄*7H₂O, 0.03 g/l FeSO₄*7H₂O,0.018 g/l MnSO₄*1H₂O, 30 g/l CaCO₃, 20 g/l glucose, 5 mg/l thiamine and50 mg/l ampicillin) and the batch is incubated for 72 hours at 37° C.After the incubation the optical density (OD) of the culture suspensionis determined with an LP2W photometer from Dr. Lange (Berlin, Germany)at a measurement wavelength of 660 nm.

The concentration of L-valine formed is then determined in thesterile-filtered culture supernatant with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column reaction with ninhydrin detection.

The result of the experiment is shown in Table 7.

TABLE 7 OD L-Valine Strain (660 nm) g/l NRRL B-12288 5.6 0.93B-12288ΔpckA 5.5 1.12

EXAMPLE 11

Construction of Deletion Mutations of the ytfP-yjfA Gene Region

The ytfP-yjfA gene region is amplified from Escherichia coli K12 usingthe polymerase chain reaction (PCR) and synthetic oligonucleotides.Starting from the nucleotide sequence of the ytfP-yjfA gene region in E.coli K12 MG1655 (SEQ ID No. 5), the following PCR primers aresynthesized (MWG Biotech, Ebersberg, Germany):

ytfP-1: 5′ - GGCGATGTCGCAACAAGCTG - 3′ (SEQ ID NO:18) ytfP-2: 5′ -CTGTTCATGGCCGCTTGCTG - 3′ (SEQ ID NO:19)The chromosomal E. coli K12 MG1655 DNA employed for the PCR is isolatedaccording to the manufacturers instructions with “Qiagen Genomic-tips100/G” (QIAGEN, Hilden, Germany). A DNA fragment approx. 1300 bp in sizecan be amplified with the specific primers under standard PCR conditions(Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications,Academic Press) with Taq-DNA polymerase (Gibco-BRL, Eggenstein,Germany). The PCR product is ligated with the vector pCR2.1TOPO (TOPO TACloning Kit, Invitrogen, Groningen, The Netherlands) in accordance withthe manufacturers instructions and transformed into the E. coli strainTOP10F′. Selection of plasmid-carrying cells takes place on LB agar, towhich 50 μg/ml ampicillin are added. After isolation of the plasmid DNA,successful cloning of the PCR product is checked with the restrictionenzymes EcoRI and NsiI.

To generate a 337 bp deletion in the yftP-yjfA region, the vectorpCR2.1TOPOytfP-yjfA is cleaved with the restriction enzymes NdeI andSspI and the DNA fragment 4.8 kbp in size is ligated, after treatmentwith Klenow enzyme.

To generate a 90 bp deletion, the vector pCR2.1TOPOytfP-yjfA is cleavedwith the enzymes NdeI and SplI and the DNA fragment 5 kbp in size isligated, after treatment with Klenow enzyme.

The E. coli strain DH5α is transformed with the ligation batches andplasmid-carrying cells are selected on LB agar, to which 50 μg/mlampicillin is added. After isolation of the plasmid DNA those plasmidsin which the mutagenic DNA sequence shown in SEQ ID No. 6 and SEQ ID No.7 is cloned are detected by control cleavage with the enzyme EcoRI. Theplasmids are called pCR2.1TOPOΔyjfA and pCR2.1TOPOΔ90bp.

EXAMPLE 12

Construction of the Replacement Vectors pMAK705ΔyjfA and pMAK705Δ90bp

The ytfP-yjfA alleles described in Example 11 are isolated from thevectors pCR2.1TOPOΔyjfA and pCR2.1TOPOΔ90bp after restriction with theenzymes SacI and XbaI and separation in 0.8% agarose gel, and ligatedwith the plasmid pMAK705 (Hamilton et al. (1989) Journal of Bacteriology174, 4617-4622), which is digested with the enzymes SacI and XbaI. Theligation batches are transformed in DH5α and plasmid-carrying cells areselected on LB agar, to which 20 μg/ml chloramphenicol are added.Successful cloning is demonstrated after isolation of the plasmid DNAand cleavage with the enzymes SacI and XbaI. The replacement vectorsformed, pMAK705ΔyjfA (=pMAK705deltayjfA) and pMAK705Δ90bp(=pMAK705delta90bp), are shown in FIG. 2 and in FIG. 5.

EXAMPLE 13

Position-specific Mutagenesis of the ytfP-yjfA Gene Region in the E.coli Strain MG442

For replacement of the chromosomal ytfP-yjfA gene region with theplasmid-coded 90 bp deletion construct, MG442 is transformed with theplasmid pMAK705Δ90bp, The gene replacement is carried out by theselection method described by Hamilton et al. (1989) Journal ofBacteriology 174, 4617-4622) and is verified by standard PCR methods(Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications,Academic Press) with the following oligonucleotide primers:

ytfP-1: 5′ - GGCGATGTCGCAACAAGCTG - 3′ (SEQ ID NO:18) ytfP-2: 5′ -CTGTTCATGGCCGCTTGCTG - 3′ (SEQ ID NO:19)The strain obtained is called MG442Δ90yjfA.

EXAMPLE 14

Preparation of L-threonine with the Strain MG442Δ90yjfA

The preparation of L-threonine by the strain MG442Δ90yjfA is tested asdescribed in Example 4. The result of the experiment is summarized inTable 8.

TABLE 8 OD L-Threonine Strain (660 nm) g/l MG442 6.0 1.5 MG442Δ90yjfA5.7 2.1

EXAMPLE 15

Preparation of L-threonine with the Strain MG442Δ90yjfAΔpckA

15.1 Preparation of the Strain MG442Δ90yjfAΔpckA

The pckA gene of the strain MG442Δ90yjfA is replaced, as described inExample 3, by the ΔpckA allele (see Example 1 and 2). The strainobtained is called MG442Δ90yjfAΔpckA.

15.2 Preparation of L-threonine

The preparation of L-threonine with the strain MG442Δ90yjfAΔpckA iscarried out as described in Example 4. The result is shown in Table 9.

TABLE 9 OD L-Threonine Strain (660 nm) g/l MG442Δ90yjfA 5.7 2.1MG442Δ90yjfAΔpckA 5.3 3.9

1. A fermentation process suitable for the preparation of a desiredL-amino acid selected from the group consisting of L-threonine,L-isoleucine, L-valine, and L-lysine, wherein the following steps arecarried out: a) fermentation of an E. coli strain in a fermentationbroth for producing the desired L-amino acid, wherein the endogenousytfP-yjfA gene region of E.coli is inactivated by one or more methods ofmutagenesis selected from the group consisting of deletion, insertionalmutagenesis due to homologous recombination, and transition ortransversion mutagenesis with incorporation of a non-sense mutation inthe ytfP-yjfA gene region, and b) concentration of the fermentationbroth to eliminate water and increase the concentration of said L-aminoacid, and c) isolation of the L-amino acid.
 2. The process according toclaim 1, wherein one or more E. coli genes selected from the groupconsisting of: (a) the thrABC operon coding for aspartate kinase,homoserine dehydrogenase, homoserine kinase and threonine synthase, (b)the pps gene coding for phosphoenolpyruvate synthase, (c) the ppc genecoding for phosphoenolpyruvate carboxylase, (d) the pntA and pntB genescoding for transhydrogenase, (e) the rhtB gene for homoserineresistance, (f) the rhtC gene for threonine resistance, and (g) the gdhAgene coding for glutamate dehydrogenase are overexpressed by increasingthe copy number or placed under a strong promoter during fermentationfor the preparation of said L-amino acids.
 3. The process according toclaim 1, wherein one or more E. coli genes selected from the groupconsisting of: (a) the tdh gene coding for threonine dehydrogenase, and(b) the mdh gene coding for malate dehydrogenase are inactivated by oneor more methods of mutagenesis selected from the group consisting ofdeletion, insertional mutagenesis due to homologous recombination, andtransition or transversion mutagenesis with incorporation of a non-sensemutation in said E. coli genes.
 4. The process of claim 1, whereinconstituents of the fermentation broth and the biomass in its entiretyor portions thereof are isolated as a solid product together with saidL-amino acid.
 5. The process according to claim 1, wherein L-threonineis produced by fermenting the E. coli strain MG442Δ90yjfA depositedunder DSM14289.